Effects of Virgin coconut oil on Testicular toxicity induced by Cadmium in rats

 

Sanjana Basavanahally Lokesh¹, Rekha D Kini², Nayanatara Arunkumar², Sneha Shetty B²,

Anupama N², Bhagyalakshmi K²

¹III MBBS Student, Kasturba Medical College, Mangalore,

Manipal Academy of Higher Education, Manipal, India.

²Department of Physiology, Kasturba Medical College, Mangalore,

Manipal Academy of Higher Education, Manipal, India.

 

ABSTRACT:

One of major threat to the environmental pollution is the contamination the by various heavy metals. Cadmium is found to have toxic effects on testis. VCO is increasingly getting the reputation of being the most versatile and healthiest antioxidant. Objective: To study the effect pre-treatment of virgin coconut oil in cadmium induced testicular toxicity in rats. Materials and methods: Institutional ethics committee clearance was obtained before beginning the experiments. Male rats of wistar strain approximately 150-200 gm body weight was used. They were housed in the central animal house under controlled conditions of temperature and light with normal rat feed and drinking water. Animals were divided into four groups with 6 rats in each group. Sperm count and sperm morphology was done. One of the testis was used for histopathological study. The data was expressed as mean ± SD. Significance of the test was set at p<0.05. Results: In the present study administration of cadmium chloride showed a significant decrease in the testicular and epididymal weight as well as sperm count and testosterone level and increase in sperm shape abnormality. Pre-treatment with Virgin coconut oil showed a significant increase in the testicular and epidydimal weight as well as sperm count and testosterone level and decrease in sperm shape abnormality Testicular section in normal control rats and Virgin treated rats showed normal features. But, in cadmium treated rats the testis showed necrosis of most of the tubules but rats pre-treated with virgin coconut oil showed that less than 25 % of the tubules had necrosis. Conclusion: In conclusion, present study showed that Cadmium administration showed a damaging effect on the testis and Virgin coconut oil showed protective role in attuning cadmium induced testicular damage

 

 

 

INTRODUCTION:

One of major threat to the environmental pollution is the contamination of the atmosphere by various heavy metals. Cadmium is one such metal which is abundant in our surrounding since industrial age¹². The main contributors of cadmium to the atmosphere are industrial and agricultural sources, cigarette smoke, lead smelting, nickel cadmium battery manufacturing and so on³.

 

Humans get exposed to cadmium either through industry or through diet³. The injurious effect of cadmium leads to various disorders like kidney and liver dysfunctions, bone diseases. reduced red blood cells toxic effects leading to alterations of the lipid profile, and testicular damage⁴. Cadmium is found to have toxic effects on testis. Many previous studied have reported that testicular toxicity of cadmium leads to male spermatogenic and steroidogenic impairment^4,5

 

Herbal medicines have been of tremendous interest since the beginning of human civilization for the treatment of all kinds of ailments⁶. Virgin coconut oil (VCO) has been used throughout the world for various health-related reasons and found to be beneficial. VCO is increasingly getting the reputation of being the most versatile and healthiest oil in the world. VCO contains fatty acids with C atomic chains, which are shorter than the fatty acid content in corn oil and soybean oil⁷ .Virgin coconut oil when consumed, turns to monolaurin, a chemical compound which is believed to increase immunity against viruses, bacteria, fungus, parasites and other pathogens⁶. There are few studies which have shown the beneficial property  of virgin oil such as anti- inflammatory, antiviral, antidiabetic and antioxidant property^8,9. Antioxidants protect the tissue from oxidative stress. Many researchers have intended at antioxidant therapy to minimise cadmium induced liver and testicular damage^10,11. There are not much studies on role of virgin coconut oil on cadmium induced hepatic and testicular toxicity. Additionally, it is likewise observed that expanding natural introduction to cadmium, presently existing word related presentation and the commonness of tobacco smoking has brought about increment in the danger of cadmium prompted lethality. So, present examination is expected to think about whether pre-treatment with virgin coconut oil will be useful in diminishing cadmium induced testicular damage

 

OBJECTIVE OF THE STUDY:

1.     To study the effects of cadmium toxicity on testis in rats.

2.     To study the effects pre-treatment of virgin coconut oil on cadmium induced testicular damage in rats.

 

MATERIALS AND METHODS:

Animals:

Institutional ethics committee clearance was obtained before beginning the experiments. Healthy adult Albino rats of wistar strain approximately 150-200gm body weight, aged 2-3 months was from the central animal house of our institution. They were housed in the central animals’ house at the laboratory under controlled conditions of temperature and light with normal rat feed and drinking water.

 

Animals were divided into the following groups with 6 rats in each group:

Group 1: Control Group (rats were injected with single dose of 0.9% NaCl intraperitoneally) and sacrificed after 15 days.

 

Group 2: (Virgin Coconut Oil treated group) rats were orally treated with VCO (of 2-ml/kg-body weight) every day for 30 days and then sacrificed.

 

Group 3: (Cadmium treated Group) where rats were injected with single dose of 1mg/kg bw cadmium chloride intraperitoneally and will be sacrificed after 15 days.

 

Group 4: (Virgin coconut oil+ cadmium treated group) where rats were pretreated with virgin coconut oil (2ml/kg bw) for 30 days and then will be injected with single dose of 1mg/kg bodyweight cadmium. Rats will be sacrificed 15 days after cadmium administration

 

After desired protocol, animals were anesthetized by giving suitable anesthesia according to the body weight. The laparotomy was performed and the reproductive organs were exposed. The testis and epididymis were weighed. The epididymis was carefully separated from the testis.

 

Sperm count: 

The epididymis was minced in 1ml of phosphate buffered saline (pH7.2) to obtain a suspension¹² The suspension was filtered through a nylon mesh. The sperm count was conducted with the filtrate in the Neubauer's chamber. Briefly, an aliquot from the suspension (up to 0.5) was taken in leukocyte hemocytometer and diluted with phosphate buffered saline up to the mark 11. The suspension was well-mixed and charged into Neubauer's counting chamber. The total sperm count in 8 squares (except the central erythrocyte area) of 1mm2 each was determined and multiplied by 5×104 to express the number of spermatozoa/ epididymis.

 

Sperm morphology test:

For the evaluation of the sperm morphology the filtrate obtained was stained with 1% eosin Y or periodic acid-Schiff's reaction and morphological defects were analyzed as explained elsewhere¹³. Briefly, the sperms in the smears were visualized under oil immersion objectives and any abnormalities of either heads or tails were noted. The microcephaly, which was also a type of head abnormality, and cephalo-caudal junction defects (CC), which were a type of tail defects have been classified separately. Two hundred sperms were screened for each animal and total abnormality was expressed as incidence / 200 sperms/animal.

.

Preparation of Virgin Coconut oil.

Virgin coconut oil (VCO) was separated from the coconut as follows: The fresh coconut meat was shredded and then cold-pressed to make coconut milk. The coconut milk was fermented for 48 hours, whereupon the oil naturally separates out from the milk, producing crystal clear oil. This unique process means that the pure oil will not have a coconut taste or odour. The virgin coconut oil was filtered and kept in a refrigerator⁷.

 

Statistical analysis:

The data was expressed as mean ± SD from 6 animals per group. Analysis was done by students t test. Statistical package SPSS version 17.0 will be used to do the analysis. Significance of the test was set at p<0.05.

 

RESULTS:

1. Effects of virgin coconut oil pre-treatment on teticular damage induced by Cadmium:

In the present study administration of 1mg/kg bw cadmium chloride showed a significant decrease in the testicular and epididymal weight. Pre-treatment with Virgin coconut oil showed a significant increase in both the testicular and epidydimal weight (Gr. IV) compared to cadmium treated group (Gr. III) (Table I)

Sperm count (P<0.001) was significantly low in cadmium treated group compared to normal control group (Table 1). Pre-treatment with Virgin coconut oil showed a significant increase in the sperm count compared to cadmium treated rats.

 

The level of testosterone was significantly low in cadmium treated group and pre-treatment with virgin coconut oil showed an increase in testosterone level compared to Cadmium treated group(P<0.05) (Table1)

 

 

Table 1: Effect of pre-treatment with virgin coconut oil on cadmium chloride induced damage on testicular weight, epididymal weight and sperm count and testosterone level. Values are expressed as Mean ± SD, n=6 in each group.

Parameters	Group I	Group II	Group III	Group IV
Sperm Count	924.46±7.90	918.74± 8.14	380.72±9.32***	748.82±6.15♦♦♦
Epididiyal weight (gm)	0.471± 0.09 0	0.468±0.07 0	0.289±0.09***	0.456±0.07♦♦♦
Testicular weight (gm/100gm BW)	0.678±0.05	0.617±0.03	0.415±0.07***	0.538±0.08♦
Testosterone level (ng/ml)	1.57±0.26	1.28±0.04	0.92±0.06***	1.21±0.29♦

***P<0.001when Gr.III is compared to Gr.I and Gr.II. ♦♦♦P<0.001, Gr.IV compared to Gr.III. ♦P<0.05 GR. IV compared to Gr.III

 

Table 2: Effect of virgin coconut oil treatment prior to cadmium chloride administration on sperm morphology in rats; Mean ± SD, n=6 in each group.

Groups	Normal	HA	TA	MC	CC	Total
Group I	163.83 ± 1.48	13.67 ± 0.52	10.50 + 0.55	0.66 + 0.52	2.33 + 0.02	26.50 ± 1.20
Group II	160.67 ± 1.44	13.12 ± 0.42	11.01 ± 0.38	0.75 ± 0.64	2.84 ± 0.04	27.72 ± 5.99
Group III	120.33 ± 1.55***	34.67 + 2.66 **	33.17 + 1.28 *	2.50 ± 0.34*	4.17 + 0.75***	75.17 ± 5.46***
Group IV	154. 74 ± 1.36♦♦♦	14.36 + 0.89♦♦	11.36 + 1.03♦	2.13 + 2.03♦	2.78 + 1.36♦♦♦	34.79♦♦♦

HA – Head abnormality, TA – Tail abnormality, MC – Microcephaly, CC – Cephalocaudal junction

***p<0.001, **P< 0.01, *P<0.05 Gr.III compared to Gr.I and Gr.II. ♦♦♦P<0.001, ♦♦P<0.01, ♦P<0.05 Gr.IV compared to Gr.III

 

A significant increase in total sperm shape abnormality was recorded in animals exposed cadmium chloride over control groups (P<0.001) (Table 2). Results of the present study showed a significant decrease in sperm shape abnormality in rats pre-treated with virgin coconut oil prior to cadmium administration compared cadmium treated rats (Table II).

 

Histological study:

Testicular section in Normal control rats (Gr.I) and Virgin treated rats showed normal features (Figure:1 and 2). But, in cadmium treated rats (GrIII) the testis showed necrosis of most of the tubules (Figure:3). The photomicrograph of the rats pre-treated with virgin coconut oil (Gr.IV) showed that less than 25 % of the tubules showed necrosis (Figure:4)

 

 

Fig. 1 Photomicrograph of rat testis shows normal histological features in the normal control group (Group I) (Haematoxylin and eosin, × 10).

 

Fig. 2 Photomicrograph of rat testis shows normal histological features in virgin treated group (Group II) (Haematoxylin and eosin, × 10).

 

Figure 4: Photomicrograph shows less than 25% of necrosis in the seminiferous tubules of rats pre-treated with virgin coconut oil prior to cadmium administration (Gr. IV) (Haematoxylin & eosin, × 10).

 

Figure 3: Photomicrograph shows necrosis in more than 75% of tubules in rats treated with 1mg/kg bw cadmium (Gr.III) (Hematoxylin & Eosin X10)

 

DISCUSSION:

Cadmium (Cd) has been recognized as one of the most toxic heavy metals and an environmental pollutant that affect the testis.¹⁴. According to Toxic Substances and Disease Registry, Cd is ranked the 7^th toxic metal. It has been well established that cadmium having a long biological half-life of approximately 20-40 years, accumulates in the human body and causes toxicity, giving way to impairment of the reproductive organs of adults, liver, kidney etc¹⁵. In the present study, exposure cadmium chloride in rats resulted in decreased sperm count and increased sperm abnormalities. These toxic effects induced by cadmium are in agreement with the effects of many other toxic metal¹⁶. The testicular and epididymal weights as well as testosterone level were significantly decreased in the cadmium exposed rat compared to control group. In the cadmium treated rats, most of the germ cells might have been destroyed either due the membranous damage or macromolecular degradation incurred by ROS leading to significant decline in the sperm count and ultimately testicular and epididymal weight loss.

 

Statistically significant increase in the percentage of sperm abnormalities in the cadmium treated rats emphasizes the possibility of gene alteration in germ cells induced by ROS generated through cadmium toxicity. Cadmium compounds do not appear to damage DNA directly, but through generating ROS which apparently causes DNA breaks. Moreover available literature during past have revealed the causation gene mutation induced by heavy metals^17,18. On the contrary, testicular germ cells carrying minor gene mutations are not eliminated but are manifested as morphologically deformed sperm. Hence, formation of abnormal sperm population in the present study is very likely due to mutagenic effects of cadmium induced ROS on specific gene loci of germ cell chromosomes involved in the maintenance of normal sperm structure. The testicular germ cells might have been destroyed either due to membrane damage or macromolecular degradation incurred by ROS leading to a significant decline in sperm count increased the incidence of abnormal sperms and ultimately testicular weight loss. The result of the present study showed histological changes like decreased spermatogenesis (less than 10% of tubule) and atrophy of the tubules. Our results are in accordance with Santos et al. reported that endothelial damage of the small blood vessels, edema and hemorrhage of the rat testes can be demonstrated by using just a single parenteral dose of cadmium chloride at 2-4mg/kg¹⁹.

 

Oxidation is a chemical reaction that removals electrons from a substance to the oxidizing agent, and cause cells damage by produce free radicals. The free radicals are produced as byproducts of normal cellular metabolic processes and scavenged by enzymatic and non-enzymatic antioxidative defense system of body. In healthy organisms, production and scavenging of free radicals is balanced by antioxidant defense system of body and any imbalance in the same leads to a condition called oxidative stress^20,21. The evolutionary survival process has provided aerobic organisms with protective systems to neutralize the oxidative effect of oxygen and its reactive metabolites. These self sustained protective components are called as “antioxidant protective systems^22,23. Antioxidants do their function either by quenching of electron mobility, scavenging free radicals or by breaking free radical chain reaction. Some recent studies have shown that virgin coconut oil (VCO) has varied degree of beneficial properties such as antiviral, antibacterial, anti-fungal, anti-inflammatory, antidiabetic, antiobesity, antiulcerogenic, analgesic and antipyretic, and antioxidant properties^24,25,26 Arunima et al in their study found that virgin coconut oil has a beneficial role in improving of virgin coconut oil on cadmium toxicity is very antioxidant status and hence preventing lipid and protein oxidation²⁷. In the present study, pre-treatment with virgin coconut oil prior to cadmium administration showed a significant increase in testicular and epididymal weight as well as testosterone level compared cadmium treated rats. Rats which we pre-treated with virgin coconut oil prior to cadmium administration also showed the normal sperm count and sperm morphology

 

CONCLUSION:

In conclusion, present study showed that Cadmium administration showed a damaging effect on the testis and Virgin coconut oil showed protective role in attuning cadmium induced testicular damage.

 

ACKNOWLEDGMENT:

We thank ICMR for accepting this project for STS 2019-20.

 

CONFLICT OF INTEREST:

Nil.

SOURCE OF FUNDING:

ICMR STS Project Fund.

 

REFERENCES:

1.   Jarup L, Akesson A. Current status of cadmium as an environmental health problem. Toxicology and Applied Pharmacology. 2009; 238(3): 201-08.

2.   Santhosh Kumar R, Asha Devi. S. Lead Toxicity on Male Reproductive System and its Mechanism: A Review. Research J. Pharm. and Tech. 2018; 11(3): 1228-1232.

3.   Adams RG. Manufacturing process, resultant risk profiles and their control in the production of nickel-cadmium(alkaline) batteries. Occup Med.1992; 42: 101-6

4.   Ellis KJ, Cohn SH, Smith TJ. Cadmium inhalation exposure estimates their significance with respect to kidney and liver cadmium burden. J Toxicol Environmental Health.1985; 15: 173-81

5.   Akinloye O, Arowojolu AO, Shittu OB, Anetor JI. Cadmium toxicity:A possible cause of male infertility in Nigeria. Reprod Biol. 2006; 6(1): 17-30.

6.   Nimbalkar V.V., Pansare P.M., Nishane B.B. Screening Methods for Hepatoprotective Agents in Experimental Animals. Research J. Pharm. and Tech. 8(12): Dec., 2015; Page 1725-1732

7.   Tristiana Erawati, Dewi Melani Hariyadi, Noorma Rosita, Tutiek Purwanti. The Anti-inflammatory Activity of p-methoxycinnamic acid (PMCA) in the Nanostructured lipid carrier (NLC) system using combinations of solid lipid, beeswax-oleum cacao and liquid lipid, Virgin Coconut oil (VCO). Research J. Pharm. and Tech 2019; 12(8): 3619-3625

8.   Hew KW, Ericson WA, Welsh MJ. A single low cadmium dose causes failure of spermiation in the rat. Toxicology and Applied Pharmacology. 1993; 121(1): 1521.

9.   S. Intahphuak, P. Khonsung, and A. Panthong, “Antiinflammatory, analgesic, and antipyretic activities of virgin coconutoil, ”Pharmaceutical Biology. 2010:.48, 151–157.

10. Z. A. Zakaria, M. N. Somchit, A. M. Mat Jais, L. K. Teh, M. Z. Salleh, and K. Long, “In vivo anti nociceptive and anti inflammatory activities of dried and fermented processed virgin coconut oil,” Medical Principles and Practice. 2011: l., 231–236.

11. Patra RC, Swarup D, Senapati SK. Effects of cadmium on lipid peroxides and superoxide dismutase in hepatic, renal and testicular tissue in rats. Vet Human,1999; Toxicol 41: 65–67

12. K. Narayana, U.J.A. D’Souza, K.P.S. Rao 2002. Effect of ribavirin on epididymal sperm count in rat, Indian J. Physiol. Pharmacol: 46(1): 97–101.

13. K. Narayana, U.J.A. D’Souza, K.P.S. Rao 2002. Ribavirin induced sperm shape abnormalities in Wistar rat, Mutat. Res: 513 (1–2): 193–196.

14. Santhosh Kumar R, Asha Devi. S. Lead Toxicity on Male Reproductive System and its Mechanism: A Review. Research J. Pharm. and Tech. 2018; 11(3): 1228-1232

15. Siu E, Mruk D, Porto C, Cheng C. Cadmium-induced testicular injury. Toxicology and Applied Pharmacology. 2009; 238(3): 240-249

16. Ebru Beytut. Mesut Aksakal. 2002. The effect of long term supplement of dietary cadmium on lipid peroxidation and the antioxidant system in the liver and kidneys of rabbits. Turk J Vet Anim Sci: 26: 1055-1060

17. Reid TM, Daniel IF, Lawrence AL. 1994. Mutagenesis by metal-induced oxygen radicals. Environ Health Prespect: 102 (suppl 3): 57–61.

18. Acharya UR, Das SS, Mishra M. 2002. Role of vitamin C and E on sperm abnormality and sperm count in cadmium treated swissmice. Cytologia: 67: 47–52.

19. Santos FW, Oro T, Zeni G, Rocha JBT. Do Nascimento PC and Nogueira CW. Toxicol Lett 2004; 152: 255-63.

20. Vandana Gautam, Dhriti Kapoor, Saroj Arora, Renu Bhardwaj. Characterization and Antioxidant Activity of oil Extract of a Gymnosperm - Araucaria cunninghamii Aiton ex D. Don. Research J. Pharm. and Tech. 2016; 9(7): 875-879.

21. Pavithra. S, N. Banu. Free Radical Scavenging Activity and Total Antioxidant Capacity of Tin Chlorophyllin from Morinda citrifolia L. Research J. Pharm. and Tech. 2017; 10(2): 453-455.

22. Davis KJA. 1986. Intracellular proteolytic systems may function as secondary antioxidant defences: a hypothesis. Free Radical Biol. Med: 2: 155-173.

23. Rekha D. Kini, Tripathi Y., C.V. Raghuveer, Sheila R. Pai. Effect of Vitamin E Pre-Treatment on Histopathological Changes on Rat Testis Following Cadmium Chloride Administration. Research J. Pharm. and Tech. 6(8): August 2013; Page 874-877.

24. Heffner JE, Repine JE. 1989. Pulmonary strategies of antioxidant defense. Am. Rev. Respir: 140: 531-554

25. Dahliatul, Qosimah, Ma. Asuncion, G.B, Aulanni’ am, A, Agri, K.A, Indah, A.A. Effect of Citrus acidity on profile of fatty acid in Virgin Coconut Oil (VCO). Research J. Pharm. and Tech 2020; 13(2): 791-794.

26. Smitha Sammith Shetty, Darrien Ray Quays, Justina John, Ng See Yee, Daneshwary a/p Appu, Lim Meng Tze, Tee Shin Chuen, Ahaliaa a/p Sachchithanantham, Sacha Miriam Augustus. Effect of Oil pulling on Oral Health- A Microbiological Study. Research J. Pharm. and Tech 2019; 12(1): 01-04.

27. Arunima S, Rajmohan T. Effect of virgin coconut oil enriched diet on the antioxidant status and paraoxonase 1 activity in ameliorating the oxidative stress in rats - a comparative study. Food Funct.2013; 4: 1402

 

 

Received on 05.06.2020           Modified on 30.08.2020

Accepted on 15.10.2020         © RJPT All right reserved